Pardee et al. Journal for ImmunoTherapy of Cancer (2015) 3:32 DOI 10.1186/s40425-015-0077-x

RESEARCH ARTICLE Open Access

Route of antigen delivery impacts theimmunostimulatory activity of dendriticcell-based vaccines for hepatocellularcarcinoma

Angela D. Pardee1, Hiroshi Yano1, Aliyah M. Weinstein1, Aaron A. K. Ponce1, Alexander D. Ethridge1,Daniel P. Normolle2, Lazar Vujanovic1,4, Gerald J. Mizejewski6, Simon C. Watkins7 and Lisa H. Butterfield1,3,4,5*

Abstract

Background: Dendritic cells (DC) are uniquely equipped to capture, process, and present antigens from theirenvironment. The context in which an antigen is acquired by DC helps to dictate the subsequent immuneresponse. Cancer vaccination promotes antitumor immunity by directing an immune response to antigensexpressed by tumors. We have tested the tumor-associated antigen alpha-fetoprotein (AFP) as an immunotherapytarget. The majority of hepatocellular carcinomas (HCC) upregulate and secrete this oncofetal antigen.

Methods: To develop cancer vaccines for HCC capable of promoting potent tumor-specific T cell responses, wetested adenovirally-encoded synthetic AFP, with or without its signal sequence, as well as protein forms of AFP andcompared intracellular routing and subsequent antigen-specific CD8+ and CD4+ T cell responses.

Results: Surprisingly, the secreted form of antigen was superior for both CD4+ and CD8+ T cell activation. We alsoexamined the mechanism through which AFP protein is endocytosed and trafficked in human DC. We identify themannose receptor (MR/CD206) as the primary uptake pathway for both normal cord blood-derived AFP (nAFP) andtumor-derived AFP (tAFP) proteins. While in healthy donors, nAFP and tAFP were cross-presented to CD8+ T cellssimilarly and CD4+ T cell responses were dependent upon MR-mediated uptake. In HCC patient cells, tAFP wasmore immunogenic, and CD4+ T cell responses were not MR-dependent.

Conclusions: Secreted, cytoplasmically retained, and endocytosed forms of AFP utilize unique uptake andprocessing pathways, resulting in different immunologic responses from the induced antigen-specific CD4+ andCD8+ T cells and between healthy donors and HCC patients. Collectively, these data elucidate pathways ofspontaneous and induced anti-tumor immunity in HCC patients to this secreted antigen.

Keywords: Alpha-fetoprotein, Dendritic cells, Hepatocellular carcinoma, Adenovirus

BackgroundDC play a key role in initiating the adaptive immune re-sponse by sampling antigens from their environmentand presenting them to lymphocytes. The physiologiccues received by DC as antigen is taken up impact pro-cessing and presentation and help to shape subsequentlymphocyte responses [1, 2]. DC were shown initially to

* Correspondence: butterfieldl@upmc.edu1Departments of Medicine, Pittsburgh, PA 15261, USA3Departments of Surgery, Pittsburgh, PA 15261, USAFull list of author information is available at the end of the article

© 2015 Pardee et al. This is an Open Access a(http://creativecommons.org/licenses/by/4.0),provided the original work is properly creditedcreativecommons.org/publicdomain/zero/1.0/

utilize both (macro)pinocytosis and the mannose receptor(MR/CD206), a C-type lectin receptor (CLR), to take upand concentrate model antigens like horseradish peroxid-ase and mannosylated bovine serum albumin (BSA) [3].Scavenger receptors, CLRs, heat shock proteins, and Fcreceptors are also antigen uptake mechanisms used by DC[4]. Endocytosis of the model protein antigen ovalbuminvia scavenger receptors promotes lysosomal degradationof ovalbumin, MHC II-restricted presentation, and activa-tion of CD4+ T cells. In contrast, uptake via MR results inenhanced cross-presentation of antigen to CD8+ T cells

rticle distributed under the terms of the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium,. The Creative Commons Public Domain Dedication waiver (http://) applies to the data made available in this article, unless otherwise stated.

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[5], which was recently shown to involve Sec61 mediatedtransport between endosomes and the cytosol [6]. Add-itional studies suggest that antigen uptake by receptors thatfacilitate early endosomal routing and accumulation of anti-gen in endosomes, rather than lysosomal delivery and pro-teolytic digestion, should promote cross-presentation,prolonged antigen presentation and subsequently inducesuperior CD8+ T cell activation, an essential component ofanti-tumor immunity [7, 8].Alpha-fetoprotein (AFP) is the most abundant serum

protein in the fetus, reaching levels of up to 3 mg/ml infetal blood [9]. It is transcriptionally repressed shortlyafter birth, and normal adult levels are between 1–20 ng/ml. The reappearance of AFP in the circulation ofadults is associated with liver regeneration, hepatitis,chronic liver diseases and malignant growth, includinghepatomas and teratomas [10]. Serum assays of circulat-ing AFP play an important role in the diagnosis of hepa-tocellular carcinoma (HCC) and monitoring responsesto treatment. In contrast to fetal AFP, tumor-secretedAFP is differentially glycosylated [11, 12]. A fucosylatedvariant, known as AFP-L3, is the major glycoform inHCC patient serum and is associated with poor outcome[13]. The uptake of AFP by multiple cell types, includingepithelial cells, activated lymphocytes and tumor cells,has been observed [14–17], although a specific cell sur-face receptor has not yet been definitively identified [17].Given its common over-expression by the majority of

HCC tumors, AFP represents an attractive target for im-munotherapy [18]. It has been demonstrated that bothCD4+ and CD8+ T cells can recognize AFP epitopespresented by dendritic cells (DC) [19]. Our group ob-served that CD8+ T cells from healthy donors canrespond to four dominant and ten subdominant HLA-A*0201-restricted peptides in vitro [20]. At least threeclinical trials have tested AFP-based vaccine regimens: i)four immunodominant HLA-A*0201-restricted AFPpeptides emulsified in Montanide adjuvant [21], ii) AFPpeptide-pulsed autologous DC [22], and iii) a DNA-prime/adenovirus (AdV)-boost genetic immunization[23]. Although no objective clinical responses were ob-served in the small numbers of vaccinated patients,AFP-specific T cell responses were either developed orexpanded in the majority of patients. The association be-tween AFP secretion and poor clinical outcome, HCCstemness [24] and tumor growth rate supports furthertesting of AFP as an immunogenic tumor-associatedantigen target. Because of the inherent variability in hu-man self-tumor antigen responses and the small size ofmost cancer vaccine clinical trials, it is not yet clear howto load DC with antigen optimally for CTL induction.Clinical trials continue to utilize a wide array of antigensources and uptake pathways to attempt to promoteantitumor immunity. It is also increasingly clear that

there is considerable tumor-immune crosstalk before tu-mors become clinically evident, and many patients havespontaneous immune responses to tumor antigens with-out vaccination or other therapy.In this study, we examined different forms of AFP

antigen to identify how the antigen is taken up, proc-essed, and presented by DC. By investigating the fetaland tumor-induced immunity to this secreted antigenand examining the subsequent impact on T cell re-sponses, we inform the design of future vaccinationstrategies targeting this oncofetal antigen.

Results and discussionAdV-transduction induces partial maturation of DCWe have previously utilized adenoviral vectors forgenetic engineering of DC due to their ability to ex-press full length antigens within DC and positively im-pact some aspects of DC function [25–29]. To furthercharacterize the maturation effects of AdV on DC, wefirst transduced healthy donor (HD) DC with an AFP-encoding AdV (AdVhAFP) and monitored the expres-sion of several maturation markers over the course of3 days. Compared to immature DC (iDC) and LPS/IFN-γ-matured DC (mDC), AdV-transduced DCexhibited intermediate expression levels of antigenpresentation molecules (HLA-ABC, HLA-DR) andcostimulatory molecules (CD40, CD83, CD80, CD86)(Fig. 1a). We also analyzed expression of the endocyticreceptors MR and CD36 following AdV-transduction(Fig. 1b). Unlike mDC, which highly downregulatethese receptors, AdV-transduced DC express levelssimilar to iDC, suggesting that AdV infection does notcompromise the endocytic function of DC.

Adenovirally-expressed AFP localizes to the Golgiapparatus and related compartments in DCTo determine the intracellular expression patterns ofadenovirally-expressed AFP, DC were transduced for3 hr and AFP localization was examined by fluores-cent microscopy for 24, 48, or 72 hr post-infection.Throughout the observation period, the AFP trans-gene was detected almost exclusively in the peri-nuclear space (Fig. 2). Adenovirally-expressed AFP isonly transiently present in early endosomes (EEA-1)at 24 h, and not detected in late endosomes/lyso-somes (LAMP-1), or the endoplasmic reticulum(KDEL). Some colocalization was observed withERGIC-53 (ER-Golgi intermediate complex), a com-partment which has been implicated in cross presen-tation [30]. However, the AFP expressed initially inthe cytoplasm from the AdV construct colocalizesextensively with Golgi (golgin-97) and trans-Golgi

A

B

Fig. 1 Phenotype of AdV-transduced DC. a and (b) Immature DC (iDC) from healthy donors (n = 3) were left untreated, matured with LPS/IFN-γ(mDC), or transduced with AdVhAFP, and then cultured in DC media for 24, 48, or 72 hr. Cells were stained for (a) antigen presentation andcostimulatory markers and (b) endocytic receptors, and analyzed by flow cytometry. Mean fluorescence intensity (MFI) is reported asthe mean ± SD

Pardee et al. Journal for ImmunoTherapy of Cancer (2015) 3:32 Page 3 of 16

(TGN46) compartments, consistent with the routingof secreted proteins through the Golgi apparatus forsubsequent release [31].

Unlike native AFP, adenovirally-encoded eGFP-AFP isretained in the cytoplasm of DCNative human AFP expressed by adenovirus (AdVhAFP) issecreted by cells due to a 19 amino acid secretion signal se-quence at the N-terminus of AFP [32]. An alternative

construct was created in which eGFP was fused to the N-terminus of AFP. Because of the proximity of eGFP to theN-terminus, this alteration masked the signal sequence andresulted in eGFP-AFP accumulation in the cytoplasm ofDC. Indeed, while the adenovirally-expressed native AFPlocalizes primarily to the perinuclear space (Fig. 3a, leftpanel), the adenovirally-expressed eGFP-AFP fusion proteinis detected throughout the cytoplasm (Fig. 3a, middlepanel). DC were also transduced with AdVTyrosinase,

EEA-1

LAMP-1

ERGIC-53

golgin-97

TGN46

24hr 48hr 72hr

KDEL

actin

Fig. 2 Intracellular trafficking of adenovirally-expressed AFP. DC were transduced with AdVhAFP at MOI 2000 for 3 hr, and then cultured in DCmedia for 24, 48, or 72 hr. Cells were then fixed and stained for AFP (green), actin, EEA-1 (early endosomes), LAMP-1 (late endosomes/lysosomes),KDEL (endoplasmic reticulum/ER), ERGIC-53 (ER-Golgi intermediate complex), golgin-97 (Golgi), and TGN46 (trans-Golgi network) (all in red), asdescribed in Materials and Methods. All images are representative of three independent experiments performed and were taken using a63x objective

Pardee et al. Journal for ImmunoTherapy of Cancer (2015) 3:32 Page 4 of 16

A

AFPeGFP

ActinAFP

ActinTyrosinase

AdVeGFP-AFP AdVTyrosinaseAdVhAFP

B

Fig. 3 Adenovirally-expressed AFP, but not eGFP-AFP, is secreted by transduced DC. a DC were transduced with AdVhAFP (left panel), AdVeGFP-AFP (middle panel), or AdVTyrosinase at MOI 2000 for 3 hr. After 48 hr, cells were fixed and stained for AFP (left and middle panels), actin (leftand right panels), or Tyrosinase (right panel). Antibody staining of AdVeGFP-AFP-transduced cells with an anti-AFP antibody reveals that AFP andGFP colocalize, as expected. Representative images from three independent experiments are shown. Images were taken using a 63x objective.b DC from healthy donors (n = 3) were transduced as above and cultured in DC media for 24, 48, or 72 hr. Supernatants were analyzed by AFPELISA. Data are reported as the mean ± SD

Pardee et al. Journal for ImmunoTherapy of Cancer (2015) 3:32 Page 5 of 16

which encodes the cytoplasmically expressed protein Tyro-sinase (Fig. 3a, right panel) for comparison. As anticipated,AdV-driven Tyrosinase and eGFP-AFP displayed similar,cytoplasmically-localized expression patterns.Clinical laboratory and ELISA assays were employed to

determine the concentration of AFP secreted into the su-pernatants of DC transduced with AdVhAFP or AdVeGFP-AFP. While native AFP is secreted at high levels over threedays post-transduction, eGFP-AFP is undetectable in thesesupernatants (Fig. 3b), further confirming that the additionof eGFP on the AFP N-terminus disrupts and inactivatesthe secretion signal.

T cell responses in HD and HCC patients are differentiallyinduced by secreted and cytoplasmically-retained AFPconstructsWe hypothesized that the presence or absence of theAFP secretion signal would impact the ability of trans-duced DC to activate AFP-specific T cells. Healthy donorPBMC were stimulated with autologous DC transduced

with either AdVhAFP or AdVeGFP-AFP, and CD8+ Tcells were analyzed 23 days later for cytokine productionupon recognition of three immunodominant, HLA-A2-restricted AFP peptides (Fig. 4a). Four of the 8 testeddonors expanded detectable AFP-specific T cells underthese short-term stimulation conditions. CD8+ T cell re-sponses against AFP137 and AFP158 were superior in theAdVhAFP group, whereas both AdVhAFP and AdVeGFP-AFP generated AFP325-specific responses, indicating someepitope specific presentation differences between the twoconstructs. Neither group was statistically significantly su-perior (global F-test, p = 0.15). We also measured TNF-αand IL-2 production by AFP-specific CD4+ T cells inhealthy donors. Again, DC transduced with the secretedAdVhAFP construct generated higher frequency helper re-sponses than the cytoplasmically-retained AdVeGFP-AFPconstruct (Fig. 4b) although statistical significance was notreached between the groups (TNF p = 0.25, IL-2 p = 0.13).We next tested the T cell stimulatory activity of these

AdVs in PBMC from AFP-positive HCC patients. Four

A

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= HD1= HD2= HD3= HD4

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= A2= A4= B3

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αα α

α

αα

αα

Fig. 4 Ability of AdV-transduced DC to induce AFP-specific T cell responses. a and (b) DC from HLA-A2+ HDs (n = 4) were transduced with AdVhAFPor AdVeGFP-AFP at MOI 2000 for 3 hr, then co-cultured with autologous PBMC for 12–13 days. a CD8+ T cells were restimulated with autologous DC(loaded with AFP as in the initial stimulation) for an additional 10d, and then analyzed for AFP-specific intracellular TNF-α production against threeimmunodominant HLA-A2-restricted peptides. b CD4+ T cells were collected after the initial stimulation and analyzed for AFP-specific intracellularcytokine production. (C and D) DC from HLA-A2+ HCC patients (n = 3–4) were transduced with AdVhAFP or AdVeGFP-AFP at MOI 2000 for 3 hr, thenco-cultured with autologous PBMC for 12–13 days. c CD8+ T cells were collected and analyzed for AFP-specific intracellular TNF-α production againstthree immunodominant HLA-A2-restricted peptides. d CD4+ T cells were collected and analyzed for AFP-specific intracellular cytokine production. Foreach panel, the solid lines represent the mean value. *, P < 0.05

Pardee et al. Journal for ImmunoTherapy of Cancer (2015) 3:32 Page 6 of 16

of the 6 HCC PBMC samples expanded detectable AFP-specific T cells. Unlike what we observed in healthydonors, DC transduced with the cytoplasmically-retainedAdVeGFP-AFP induced superior or equivalent CD8+ Tcell responses (with some epitope-specific differences)versus the AdVhAFP group (Fig. 4c). AFP137 differencesapproached significance (p = 0.06) and AFP325 differenceswere highly significant (p < 0.0001). HCC patient CD4+ Tcells were also efficiently activated by both constructs

(Fig. 4d), suggesting that in individuals with prior exposureto tumor-derived AFP, but not in healthy donors, robustAFP-specific T cell responses can be induced by bothsecreted and cytoplasmically-retained AFP.

AFP protein upregulates maturation markers anddownregulates endocytic receptors on DCWe have recently shown that monocytes cultured withHCC tumor-derived AFP (tAFP), but not normal cord

Pardee et al. Journal for ImmunoTherapy of Cancer (2015) 3:32 Page 7 of 16

blood-derived AFP (nAFP), fail to fully differentiateinto DC, despite the fact that nAFP and tAFP isoformsonly differ at one carbohydrate group [33]. Here, wenext investigated the effects of nAFP and tAFP on DCthat were first differentiated before being exposed toAFP. Compared to untreated iDC, nAFP- and tAFP-treated DC expressed slightly higher levels of several anti-gen presentation and costimulatory molecules (Fig. 5a).

A

B

Fig. 5 Phenotype of AFP-loaded DC. a and (b) Immature DC (iDC) from hetAFP (10 μg/ml) in DC media for 24, 48, or 72 hr. Cells were stained for (a)receptors, and analyzed by flow cytometry. Mean fluorescence intensity (M

Together with our previous study, these data under-score the multifunctional nature of AFP: potentlysuppressive in monocytes, yet somewhat stimulatory inpreviously differentiated DC. Expression of the endocy-tic receptors MR and CD36, however, was diminished innAFP- and tAFP-treated DC (Fig. 5b), suggesting thatAFP protein negatively regulates the endocytic activityof DC.

althy donors (n = 3) were left untreated or cultured with nAFP andantigen presentation and costimulatory markers and (b) endocyticFI) is reported as the mean ± SD

A

Actin = rednAFP = greenNucleus = blue

0 min

30 min

60 min

Chase:

CChase: 0 hr 24 hr

EEA-1

LAMP-1

KDEL

golgin-97

4°37°

nAFP tAFP

B

Fig. 6 Intracellular trafficking of endocytosed AFP. a nAFP and tAFP were Alexa Fluor 488-labeled. Human monocyte-derived DC were co-cultured with AFP (10 μg/ml) for 1 hr at 4 °C or 37 °C, and analyzed by flow cytometry. b DC were co-cultured with fluorescently-labeled nAFPfor 2 hr, then chased with media alone for the indicated times. Cells were then fixed, stained for actin (described in Materials and Methods), andanalyzed by confocal microscopy. c DC were incubated with fluorescently-labeled nAFP for 2 hr, then chased with media alone for the indicatedtimes. Cells were then fixed and stained for EEA-1 (early endosomes), LAMP-1 (late endosomes/lysosomes), KDEL (endoplasmic reticulum/ER), andgolgin-97 (Golgi) (all in red). All images are representative of three independent experiments performed and were taken using a 63x objective

Pardee et al. Journal for ImmunoTherapy of Cancer (2015) 3:32 Page 8 of 16

Intracellular trafficking of endocytosed AFPTo understand the dynamics of AFP internalization byDC, we first labeled nAFP and tAFP with Alexa Fluor488 and verified by flow cytometry that both proteinswere efficiently taken up by DC (Fig. 6a). AFP endocyto-sis was further examined by fluorescent microscopy.After a one hour pulse of DC with fluorescently-labelednAFP, cells were chased with media alone for 0, 30, and60 min (Fig. 6b). Immediately following the AFP antigenpulse, endocytic pockets containing nAFP are observedthroughout the cytoplasm. This was further examinedwith live cell imaging which shows that AFP is detect-able within DC within 30 s (data not shown). By 30 minpost-pulse, nAFP exhibits a perinuclear localization.Similar trafficking patterns were observed with tAFP(data not shown). A recent report [30] identified a peri-nuclear endosomal recycling compartment (ERC) in DCwhich contain high levels of MHC class I molecules, and

which played a role in cross-presentation. We stained ourAFP loaded DC for MHC class I and did not see colocaliza-tion (not shown), indicating that the peri-nuclear AFP isnot in an MHC-loaded ERC. Colocalization of nAFP inintracellular compartments was examined after a one hourpulse and 0 or 24 h chase (Fig. 6c). Immediately followingthe pulse, nAFP colocalized with early endosomes (EEA-1),but not late endosomes/lysosomes (LAMP-1), endoplasmicreticulum (KDEL), or Golgi (golgin-97). At 24 h post-pulse,nAFP exhibited extensive colocalization with LAMP-1,consistent with a classical MHC Class II processing/presen-tation paradigm. Similar trafficking patterns were observedwith tAFP (data not shown).

DC endocytose AFP primarily via the mannose receptorAlthough an AFP cell surface receptor has been partiallycharacterized biochemically [17], it has not been defini-tively cloned. Several molecules have been proposed as

A

MR:99.9%

DC-SIGN:99.4%

SR-A1:21.6%

LOX-1:2.1%

CD36:99.9%

SR-B1:29.6%

C-type lectin receptors Scavenger receptors

B

C

MR = rednAFP = green

D

CD36 = rednAFP = green

Fig. 7 DC endocytose AFP primarily via the mannose receptor. a DC were stained for flow cytometric analysis. Red histogram represents endocyticreceptor staining; black histogram represents isotype control staining. Data from one representative healthy donor (of three total HD tested) is shown.b DC were pre-treated with inhibitors for 30 min, and co-cultured with fluorescently-labeled nAFP (top panels) or tAFP (bottom panels) for an additional1 hr. Percent inhibition was calculated based on untreated control cells. Columns, mean of four HD; bars, standard deviation. c and (d) DC were incubatedwith fluorescently-labeled nAFP for 2 hr, then fixed and stained for MR (c) or CD36 (d). All images are representative of three independent experimentsperformed and were taken using a 63x objective

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putative receptors for AFP, including the mannose re-ceptor (MR/CD206) and several scavenger receptors(SR-A1, LOX-1, CD36, SR-B1) [34]. We first examinedDC for expression of these receptors. As depicted inFig. 7a, DC express high levels of MR, DC-SIGN, andCD36. To identify the mechanism(s) of AFP uptake, DCwere pretreated with inhibitors against pinocytosis(DMA), CLR-mediated endocytosis (mannan), and scav-enger receptor-mediated endocytosis (polyinosinic acid;poly I). Cells were then pulsed with fluorescently-labelednAFP and tAFP and uptake inhibition calculated(Fig. 7b). While the blockade of receptor-independent

pinocytosis by DMA displayed moderate suppressiveactivity, uptake of nAFP and tAFP was substantiallyreduced by mannan and poly I, indicating that CLR- andscavenger receptor-mediated endocytosis accounts forthe majority of AFP protein internalization. AFP intern-alization by DC was not abrogated by GalNAc, which in-hibits galactose-specific CLRs (i.e. MGL) (data notshown). We also targeted individual CLRs and scavengerreceptors using blocking antibodies. Of those tested,only the MR blocking antibody demonstrated suppres-sive activity, suggesting that the primary mechanism ofAFP uptake in DC is through MR-mediated endocytosis.

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We further analyzed the role of MR in AFP uptakeusing fluorescent microscopy. Immediately after DCwere pulsed with fluorescently-labeled nAFP, endocyticpockets containing MR and nAFP, together and co-localized, were observed throughout the cytoplasm ofDC, further supporting the role of MR in AFP endocyto-sis (Fig. 7c). Similar trafficking patterns and MR co-localization were observed with tAFP (data not shown).No colocalization was observed, however, between endo-cytosed nAFP and the scavenger receptor CD36(Fig. 7d).Because HCC cells are also capable of taking up AFP

[35], endocytosis of nAFP and tAFP by the HCC cell lineHepG2 was also assessed. HepG2 cells take up compar-able levels of both nAFP and tAFP (Additional file 1:Figure S1A), although to a lesser degree than DC(Fig. 6a). We also confirmed that AFP is internalized byHepG2 cells and not simply bound to the cell surface(Additional file 1: Figure S1B). Unlike DC, HepG2 cellsdo not express CLRs, but express high levels of CD36and moderate levels of LOX-1 and SR-B1 (Additional file1: Figure S1C). By utilizing the same uptake inhibitionstrategy, we demonstrate that AFP uptake occurs pri-marily through pinocytosis and scavenger receptors, incontrast to DC (Additional file 1: Figure S1D).

AFP-specific T cell responses induced by protein-loadedDC occur in both MR-dependent and –independentmannersPrevious studies by our group have shown AFP protein-loaded immature DC to be inferior to AdVhAFP-transduced DC at inducing AFP-specific CD4+ and CD8+ T cell responses [36, 37]. In this study, the AFPprotein-loaded DC were matured with LPS/IFN-γ priorto T cell stimulation to determine if a more optimizedprotein loading procedure would activate strong T cellresponses. We also wanted to assess both glycoforms ofAFP (nAFP and tAFP), as well as the significance ofMR-mediated AFP endocytosis in the activation of Tcells by DC. Healthy donor DC were pulsed with nAFPor tAFP in the presence or absence of MR blocking anti-body, matured for an additional 24 h, then co-culturedwith autologous PBMC. Several days later, CD8+ T cellswere analyzed for cytokine production in response toAFP-derived peptides (Fig. 8a). This allowed us to detectcross-presentation of the exogenously added protein,compared to the cytoplasmically delivered virallyencoded AFP tested above (Fig. 4). In general, multi-epitope CD8+ T cell responses were generated from bothnAFP or tAFP protein, which was not inhibited by MRblockade, indicating that the glycosylation state androute of uptake of AFP by DC are not critical parametersfor priming CD8+ T cells of HD. The frequencies of CD8+ T cells expanded from protein cross-presentation were

generally similar to that of cytoplasmic antigen in thematured DC although the exogenous protein loaded DCactivated significantly more AFP325 or AFP158-specific Tcells (p = 0.007). Robust HD CD4+ T cell responses,however, were almost completely abrogated by MRblockade in 3 of 4 donors (Fig. 8b), suggesting that MR-mediated routing of internalized AFP into the MHCClass II pathway is important for AFP-specific CD4+ Tcell priming. Exogenous protein yielded higher frequen-cies of CD4+ T cell responses than either virally encodedform.Protein-loaded HCC patient DC were next evaluated

for their T cell stimulatory activity. As with healthy do-nors, MR blockade did not compromise the ability ofthese DC to cross-present antigen and induce high fre-quency CD8+ T cell activation (Fig. 8c). In fact, MRblockade significantly increased the frequency of AFP158and AFP325-specific T cells stimulated by nAFP loadedDC (p = 0.02 and 0.046, respectively). While inhibitingtAFP stimulation of AFP158-specific CD8+ T cells (p =0.0008). Importantly, MR blockade had a negligible ef-fect on AFP-specific CD4+ T cell activation in HCC pa-tients (Fig. 8d). This is in contrast to the reduced CD4+

T cell responses with MR blockade in healthy donors.These data suggest that HCC patients with AFP-secreting tumors may have been previously primed bycirculating tAFP, and that our in vitro cultures are test-ing the loaded DC ability to boost these T cells. Indeed,the frequencies of AFP-specific CD4+ T cells are muchhigher (Figs. 4d and 8d) than from healthy donors(Figs. 4b and 8b). Together with our AdV-delivered anti-gen results (Fig. 4), these data show that T cells fromAFP-naïve healthy donors and AFP-experienced HCCpatients are differentially activated by alternative strat-egies and pathways of DC antigen loading.

ConclusionsNumerous cell surface receptors on DC, including MR,DEC-205, DC-SIGN, and several scavenger receptors,mediate the endocytosis of exogenous antigens. Internal-ized antigen is generally shuttled into lysosomes, whereit is subsequently degraded and presented in the contextof MHC Class II molecules. Alternatively, DC are cap-able of re-routing internalized antigen into the MHCClass I pathway via several potential mechanisms [38].This process, known as cross-presentation, is essentialfor the development of protective anti-tumor CD8+ Tcell immunity. Importantly, recent evidence suggeststhat the divergence of internalized antigen into eitherMHC Class I or Class II pathways may be dictated bythe particular cell surface receptor that was responsiblefor endocytosis of the antigen. We have recently shownthat AFP, a secreted oncofetal antigen that is over-expressed by more than half of HCC tumors, is

nAFP

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FP

tAFP +

MR

0.0

0.2

0.4

0.6

0.8

% IL

-2 p

osi

tive

**

α

α α α α

α α α α

α α α α

α α α α α α

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Fig. 8 Ability of AFP protein-loaded DC to induce AFP-specific T cell responses. a and (b) DC from HLA-A2+ HDs (n = 4) were cultured with nAFPor tAFP (at 10 μg/ml for 2 hr) with or without pre-treatment with MR blocking antibody (at 10 μg/ml for 30 min), matured for an additional 24 h,then co-cultured with autologous PBMC for 12–13 days. a CD8+ T cells were restimulated with autologous DC (loaded with AFP as in the initialstimulation) for an additional 10d, and then analyzed for AFP-specific intracellular TNF-α production against three immunodominant HLA-A2-restricted peptides. b CD4+ T cells were collected after the initial stimulation and analyzed for AFP-specific intracellular cytokine production. c and(d) DC from HLA-A2+ HCC patients (n = 3–4) were cultured with nAFP or tAFP (at 10 μg/ml for 2 hr) with or without pre-treatment with MR blockingantibody (at 10 μg/ml for 30 min), matured for an additional 24 h, then co-cultured with autologous PBMC for 12–13 days. (C) CD8+ T cells were collectedand analyzed for AFP-specific intracellular TNF-α production against three immunodominant HLA-A2-restricted peptides. (D) CD4+ T cells were collectedand analyzed for AFP-specific intracellular cytokine production. For each panel, the solid lines represent the mean value. *, P < 0.05

Pardee et al. Journal for ImmunoTherapy of Cancer (2015) 3:32 Page 11 of 16

efficiently internalized by human monocyte-derived DC.Here, to identify the endocytosis mechanism(s) respon-sible for AFP internalization and to determine the down-stream impact on T cell responses, we co-cultured DC

with fluorescently-labeled AFP in the presence or ab-sence of inhibitors of specific endocytic pathways.Pretreatment of DC with mannan, an MR inhibitorthat competitively blocks endocytosis of mannose-rich

Pardee et al. Journal for ImmunoTherapy of Cancer (2015) 3:32 Page 12 of 16

structures, abrogated AFP uptake, while similar treat-ment with polyinosinic acid, a specific inhibitor ofscavenger receptors, blocked between 50-70 % of AFPinternalization. These data are consistent with the ob-served high levels of MR and the scavenger receptorsSR-A1 and CD36 expressed by the DC. MR is alsoknown to be expressed on circulating human BDCA1+

DC [39]. Because AFP is also taken up by HCC cells,where it is thought to regulate apoptosis and steroidreceptor-mediated cell growth, we performed similarendocytosis blockade experiments using the HCC cellline HepG2. In contrast to DC, uptake of AFP byHepG2 cells was mediated by pinocytosis and severalscavenger receptors, including LOX-1 and CD36.Vaccines, including cancer vaccines, have utilized many

approaches to convey antigen to stimulatory antigen pre-senting cells. Ex vivo DC loading strategies include tumorfusion, tumor lysate loading (with a variety of tumor kill-ing strategies), purified protein loading, antibody conju-gate targeting, long peptide loading, and MHC-restrictedshort peptide epitope pulsing [40]. Each of these ap-proaches has been used in clinical trials with variable im-munologic and therapeutic outcomes [41–43]. The role ofthe antigen uptake pathway in not generally addressed inthese studies.The MR has been shown to be highly expressed on

cultured DC and to be a very efficient antigen uptake re-ceptor [44]. To improve the uptake efficiency of someantigens, conjugation of mannose groups has been testedand shown to improve uptake by DC [45]. This CD206pathway allows DC to accumulate a large amount of avariety of antigen molecules as well as microbes withmannosylated glycoproteins very quickly [3], with veryefficient receptor recycling. Depending on the antigenglycosylation state, as little as 15 min of antigen uptakecan result in maximal T cell activation [44]. Here, weshow that uptake of AFP is extremely efficient, withintracellular antigen detected immediately after pulsing.The mature, circulating form of AFP contains approxi-mately 4 % carbohydrates by weight, including N-acetyl-glucosamine (1.2 %), mannose (2.2 %), sialic acid (0.9 %)and small amounts of glucose [46]. Binding of AFP re-gions to MR were predicted in silico by an algorithm de-signed by one of us [47], with AFP 485–493 and AFP492–500 in domain 3 being critical. As we [33] andothers [48–53] have shown, AFP can have immune sup-pressive effects on DC and we have shown that a signifi-cant amount of that activity is due to low molecularmass (LMM) co-purifying molecules bound to AFP thatis internalized with the AFP. Here, we have shown thatMR-mediated uptake of both nAFP and tAFP is very ef-ficient, allowing for a quick accumulation of AFP proteinin early endosomes, which can be detected in the peri-nuclear space for at least 72 h. This uptake pathway may,

therefore, enhance the ability of the co-purifying mole-cules (known to include fatty acids, bilirubin and neop-terin [54, 55]) to be concentrated inside DC. Blockade ofCD206 reduced HD CD4+ T cell responses, suggestingthat scavenger receptor-mediated uptake does not supportefficient antigen routing for MHC class II presentation,while CD206 mediated routing through early endosomesis the most efficient route. Unexpectedly, tAFP and nAFPbehave similarly as antigen sources for CD8+ and CD4+ Tcell activation. The only significant difference we detectedwas that MR blockade improved CD8+ T cell responses inHCC patients from nAFP loaded DC. The frequencies oftAFP-induced T cells was also higher frequency in HCCpatients than for nAFP.Replication-deficient AdVs are also an established gene

delivery vehicle, capable of transducing both dividingand non-dividing cells and inducing prolonged transgeneexpression (up to 14 days) into transduced cells [56].Type 5 AdV bind to the cell surface Coxsackie-adenovirus receptor (CAR) and cell-surface integrins,leading to clathrin-mediated endocytosis. The virus-containing macropinosomes are lysed, releasing thecontents into the cytosol [57]. We found that theadenovirally-delivered cytoplasmic eGFP-AFP was dif-fusely localized throughout the cell, without any accu-mulation in the perinuclear regions. The superiority ofthe native secreted form of AFP for priming of bothCD4+ and CD8+ T cell responses in HD may relate tothe high level accumulation of antigen over several daysthroughout the ERGIC, Golgi and TGN. Our observa-tions are also similar to those described by Fukasawa etal., in which recombinant native AFP was routed intothe secretory pathway of various cell lines via the Golgi,whereas an AFP construct lacking the N-terminal secre-tion signal was retained in the cytoplasm and failed tobe secreted [58].We hypothesize that healthy donor T cells have been

exposed to nAFP, taken up via CD206 and SR pathways,and presented to CD4+ and CD8+ T cells, during fetaldevelopment through birth. This is in contrast to othertypes of self antigens, like cytoplasmically expressed pro-teins from adult tissues like melanocyte lineage antigens.HCC patients with tAFP-expressing tumors have beenfurther exposed as adults to circulating tAFP. While wedemonstrate here that both nAFP and tAFP are effi-ciently endocytosed by DC via CD206 and SRs, we re-cently demonstrated that tAFP binds to potentlyimmune suppressive molecules that can significantly im-pair DC function and T cell stimulatory activity [33].Hepatocellular carcinoma (HCC), the most common

form of liver cancer, is the second leading cause of cancer-related death worldwide [59]. Current therapies for ad-vanced HCC are marginally effective and can exacerbateunderlying liver disease. The ability of immunotherapy to

Pardee et al. Journal for ImmunoTherapy of Cancer (2015) 3:32 Page 13 of 16

elicit nontoxic, systemic, long-lived anti-tumor activitytherefore makes it an attractive area of investigation, andthere have been a few intriguing trials with improved out-comes for vaccinated HCC patients [60, 61]. Importantly, aminority of HCC tumors are spontaneously infiltrated bylymphocytes [62], and to date, HCC has not been one ofthe more responsive tumor types with checkpoint blockadewith anti-CTLA-4 or anti-PD-1 [63]. It may be critical tofirst drive a tumor antigen immune response with vaccin-ation to create a setting for response to other therapies.

MethodsReagentsHuman cord serum nAFP (Cell Sciences; purity >95 %by SDS-PAGE), and HCC cell line culture-derived tAFP(Bio-Rad; purity >95 % by SDS-PAGE) were added tocultures at 10 μg/ml. Alexa Fluor 488-conjugation ofnAFP and tAFP was performed using an Alexa Fluor488 protein labeling kit (Life Technologies). Overlappingpeptides (20-mer amino acids overlapping by 10) span-ning the AFP protein were from Mimotopes (>80 %purity). Lyophilized peptides were reconstituted inDMSO and pooled. AFP-derived peptides (AFP137–145:PLFQVPEPV; AFP158–166: FMNKFIYEI; AFP325–334:GLSPNLNRFL) were purchased from the University ofPittsburgh Peptide Synthesis Facility. E1- and E3-deletedadenoviruses encoding human AFP (AdVhAFP) and en-hanced green fluorescent protein (eGFP)-tagged AFP(AdVeGFP-AFP) were acquired from BDP/NCI-Fred-erick and the University of Pittsburgh Vector Core, re-spectively. AdVTyrosinase was also acquired from theUniversity of Pittsburgh Vector Core. Viral titers weretested using the Adeno-X Rapid Titer Kit (Clontech La-boratories) and multiplicity of infection (MOI) used toinfect DC was based on the values obtained.

Cell linesHepG2 hepatoma (ATCC #HB-8065) and T2 (HLA-A2+; ATCC #CRL-1992) cell lines were cultured in RPMI1640 medium, supplemented with 10 % fetal bovineserum, 1 % penicillin-streptomycin, and 1 % L-glutamine(all reagents from Life Technologies). Cultures weremaintained in a humidified 37 °C incubator under 5 %CO2 tension.

Isolation of PBMCsPeripheral blood mononuclear cells (PBMC) were ob-tained from 8 healthy donors (HD) and from 6 HCC pa-tients banked from an AFP peptide-pulsed DC vaccineclinical trial [22] (UPCI #04-001; UCLA IRB #00-01-026,IND BB9395; informed consent was obtained from allpatients and donors). Limited patient data are listed inAdditional file 2: Table S1. PBMC were separated from

blood using gradient centrifugation (Ficoll-Paque, GEHealthcare).

DC preparationCD14+ monocytes were isolated from PBMC using mag-netic cell sorting (Miltenyi Biotec) and cultured for 5 daysin 800 IU/ml rGM-CSF (Sargramostim; Genzyme) and500 IU/ml rIL-4 (eBioscience; purity >98 % by SDS-PAGE).Where noted, DC were matured with IFN-γ (1000 IU/ml;PeproTech) and lipopolysaccharide (LPS, 250 ng/ml;Sigma-Aldrich) for an additional 24 h prior to collection.DC were transduced with AdVhAFP, AdVeGFP-AFP, orAdVTyrosinase for 3 hr in serum-free media at MOI 2000.

Flow cytometryEndocytic receptors were stained using the followingantibodies: CD206/MR (eBioscience), DC-SIGN, CD36(both from BD Biosciences), SR-A1 (R&D Systems),LOX-1 (BioLegend), and SR-B1 (Novus Biologicals). DCand T cell phenotypes were examined using antibodiesagainst the following markers: HLA-ABC (BioLegend),CD206, CD40, CD80, CD83, IL-2 (BD Biosciences),CD4, CD8, TNF-α, IL-2, and HLA-DR (BeckmanCoulter). Data were acquired with an Accuri C6 cyt-ometer (BD Biosciences) and analyzed using CFlow Plussoftware.

Uptake inhibition assaysDC or HepG2 cells were pretreated with inhibitors inserum-free media for 30 min at 37 °C. Without washing,fluorescently-labeled protein was added at 10 μg/ml for1 hr at 37 °C. After washing, MFI of internalized proteinwas quantitated by flow cytometry. Percent inhibitionwas calculated as: [(MFI of untreated cells) – (MFI oftreated cells)]/(MFI of untreated cells) × 100 %. The fol-lowing blocking antibodies were used: CD206 (clone15–2; BioLegend), DC-SIGN (clone 120507), SR-A1(clone 351620), LOX-1 (clone 331212; all from R&DSystems), CD36 (clone 185-1G2; NeoMarkers), andSR-B1 (rabbit polyclonal; Novus Biologicals). Dimethy-lamiloride (DMA; 100 μM), mannan (300 μg/ml), andpolyinosinic acid (Poly I; 50 μg/ml) were purchasedfrom Sigma.

Confocal immunofluorescence staining and imagingDC cultured in 8-well chamber slides (Nunc) were fixed(4 % paraformaldehyde), permeabilized (0.1 % Triton X-100), and stained with rhodamine phalloidin (Life Tech-nologies) and DRAQ5 (eBioscience), to label F-actin andnuclei, respectively. The following primary antibodieswere used for staining cells: AFP (AbD Serotec), CD206(BioLegend), CD36, Tyrosinase, EEA-1, LAMP-1, KDEL,ERGIC-53 (all Santa Cruz Biotechnology), golgin-97

Pardee et al. Journal for ImmunoTherapy of Cancer (2015) 3:32 Page 14 of 16

(Life Technologies), TGN46 (Sigma), mouse and rabbitIgG isotype controls (R&D Systems), and anti-AlexaFluor 488 (to amplify the fluorescent signal of AlexaFluor 488-labeled protein; Life Technologies). The follow-ing secondary antibodies were used: goat anti-mouseAlexa Fluor 488 and 555, goat anti-rabbit Alexa Fluor 488and 555 (all from Cell Signaling Technology), donkeyanti-goat Cy3 (Jackson ImmunoResearch), donkey anti-mouse Alexa Fluor-488, and donkey anti-rabbit AlexaFluor-488 (both Life Technologies). Images were acquiredusing a Leica TCS-SL confocal microscope.

Quantification of secreted AFP by ELISASecretion of AFP and eGFP-AFP by adenovirus vector-transfected DCs was quantified using DuoSet ELISA de-velopment system for human AFP (R&D Systems) perthe manufacturer’s protocol.

T cell stimulation and cytokine production assaysAFP-loaded DC (via AdVhAFP transduction or AFPprotein pulse) from HLA-A2+ HD or HCC patientswere co-cultured with autologous monocyte-depletedPBMC at a 1:10 ratio (DC: PBMC) in RPMI 1640 plus10 % human AB serum plus pen/strep/L-G In plus20 IU/ml IL-2 and 5 ng/ml IL-7. Cultures were supple-mented with fresh media containing IL-2 and IL-7 every3–4 days. After 12–13 days, HD CD4+ T cells and HCCpatient CD8+ and CD4+ T cells were analyzed for AFP-specific cytokine production, while HD CD8+ T cellswere isolated by magnetic bead selection (MiltenyiBiotec) and restimulated with autologous DC (loadedwith AFP as in the initial stimulation) for an additional10d, and then analyzed for AFP-specific cytokine pro-duction. For CD8+ T cell intracellular cytokine staining,cells were stimulated in the presence of brefeldin A withT2 cells pre-loaded with one of three immunodominantAFP-derived peptides (AFP137–145, AFP158–166, andAFP325–334; all at 10 μg/ml). Six hours later, T cells werestained for CD8, fixed, permeabilized, and stained forintracellular TNF-α. For CD4+ T cell intracellular cyto-kine staining, cells were stimulated in the presence ofbrefeldin A with autologous DC pre-loaded with theAFP peptide pool (at 60 μg/ml). Six hours later, T cellswere stained for CD4, fixed, permeabilized, and stainedfor intracellular IL-2 and TNF-α.

Statistical analysisTo determine the statistical significance of differences inAFP antigen form and uptake pathways, Figs. 4 and 8were analyzed as follows. Mixed effects ANOVA were fitto the data, and the distributions of the residuals werevisually checked using Q-Q plots. While some of thedata demonstrated serious departures from the distribu-tional assumption, these departures were not present in

any of the analyses with statistically significant results.P values < 0.05 are considered significant and noted withasterisks in the figures.

Additional files

Additional file 1: Figure S1. HepG2 cells endocytose AFP primarily viapinocytosis and scavenger receptors. (A) nAFP and tAFP were AlexaFluor 488-labeled. HepG2 cells were co-cultured with AFP (10 μg/ml)for 1 hr at 4 °C or 37 °C, and analyzed by flow cytometry. (B) HepG2cells were co-cultured with fluorescently-labeled nAFP for 2 hr, thenfixed, stained for actin (described in Materials and Methods), andanalyzed by confocal microscopy. (C) HepG2 cells were stained for flowcytometric analysis. Red histogram represents endocytic receptorstaining; black histogram represents isotype control staining. Data fromone representative experiment (of three total) is shown. (D) HepG2 cellswere pre-treated with inhibitors for 30 min, and co-cultured withfluorescently-labeled nAFP (left panel) or tAFP (right panel) for anadditional 1 hr. Percent inhibition was calculated based on untreatedcontrol cells. Columns, mean of three independent experiments; bars,standard deviation.

Additional file 2: Table S1. HCC patient demographics.

Competing interestsADP, HY, AMW, AAKP, SCW, GJM and ADE: none to declare. LHB is co-inventorof patents covering aspects of AFP-derived peptides as targets for Tcell-mediated anti-HCC immunity.

Authors’ contributionsADP designed experiments, performed uptake, imaging, flow cytometry,transduction, ELISA, and T cell culture experiments, analyzed data and wrotethe manuscript. HY performed uptake, ELISA and imaging experiments. AMWperformed uptake and imaging experiments. AAKP performed uptake andimaging experiments. ADE performed transduction and imagingexperiments. DPN performed statistical analyses and helped write themanuscript. LV analyzed data and helped write the manuscript. GJMprovided advice about binding and uptake experiments and helped writethe manuscript. SCW helped to design and interpret the imagingexperiments. LHB conceived of the study, designed experiments, reviewedall data and wrote the manuscript. All authors read and approved the finalmanuscript.

AcknowledgmentsThis study was supported by research funding from the University ofPittsburgh Cancer Institute (UPCI) P30 CA047904 and NCI RO1 CA 138635(LHB). This project used UPCI shared resources that are supported in part byaward P30CA047904: the UPCI Immunologic Monitoring and CellularProducts Laboratory (L.H. Butterfield, Director), the University of PittsburghVector Core (A. Gambotto, Director), and the University of Pittsburgh Centerfor Biologic Imaging (S. Watkins, Director and P. Basse, Co-Director).

Author details1Departments of Medicine, Pittsburgh, PA 15261, USA. 2Departments ofBiostatistics, Pittsburgh, PA 15261, USA. 3Departments of Surgery, Pittsburgh,PA 15261, USA. 4Departments of Immunology, University of PittsburghSchool of Medicine, Pittsburgh, PA 15261, USA. 5University of PittsburghCancer Institute, Hillman Cancer Center 5117 Centre Avenue, Suite 1.27,Pittsburgh, PA 15213, USA. 6Non-paid Advisor at the Wadsworth Center, NewYork State Department of Health, Albany, NY 12201, USA. 7Department ofCell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA 15261,USA.

Received: 19 March 2015 Accepted: 24 June 2015

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